Isolation and identification of cryptosporidium parvum oocysts with continuous percoll gradients and combined alcian blue-giemsa staining

Abstract

The development of special techniques for the detection of Cryptosporidium oocysts in feces contributed significantly to our understanding of human and animal parasitoses by this organism. Garcia et al. (1983) who compared several techniques for the detection of Cryptosporidium oocysts, reported that the most effective method of concentration in their lab was flotation on a Sheather's solution (Sheather, 1923); material thus prepared did not require staining if observed with phase contrast microscopy. Few techniques for the purification of Cryptosporidium oocysts have been developed subsequently. To date, the most widely used of the newer approaches include separation on discontinuous Percoll gradients (Waldman et al., 1986; Arrowood and Sterling, 1987; Kilani and Sekla, 1987) and the use of the parasite concentrator device (FPC) (Evergreen Scientific, Los Angeles) developed by Zierdt (1984). These techniques did not yield good results due to high concentrations of contaminants in the oocyst suspensions. By testing new approaches to oocyst isolation, we found the use of Concanavalin A-Sepharose 4B to be costly, whereas separation with continuous Percoll gradients was faster, and recovered up to 80% of all oocysts present in the initial sample. Of the staining techniques tried to date, the most effective for diagnosis in fecal samples is the modified Zielh-Neelsen method by Bronsdon (1984). Fecal samples containing Cryptosporidium parvum oocysts were obtained from spontaneusly infected calves. For transport to the laboratory, the samples were mixed with an equal volume of 2.5% potassium dichromate. In the laboratory, gross remains were filtered out with nylon nets of decreasing mesh sizes (100 gm, 75 gm,